Avantatges del PGD en 1CP amb CGH

El major avantatge de la CGH és l’anàlisi fiable de pràcticament la totalitat de cromosomes, i, per tant, la detecció no únicament de les aneuploïdies més freqüents, sinó també de la resta d’aneuploïdies que podrien causar una pèrdua embrionària primerenca o una fallada d’implantació. La CGH, a més, permet una avaluació fiable de les hipohaploïdies ja que en aquest cas, la fixació del material cromosòmic no és necessària i les pèrdues artefactuals dels cromosomes no es poden produir. A més a més, la CGH permet analitzar els cromosomes en tota la seva longitud, a diferència de la FISH, en la que només s’identifiquen certes regions del cromosoma (centròmers, telòmers o determinats gens). Això ha permès detectar trencaments cromosòmics en embrions humans (Voullaire et al.

2000; Wells i Delhanty 2000), i indica que la CGH és una tècnica molt útil per detectar alteracions estructurals de novo resultants en una pèrdua o un guany de material cromosòmic.

Les optimitzacions fetes en el protocol permeten fer la CGH en unes 60 hores, corresponents aproximadament a 13 hores per al protocol de DOP-PCR-CGH i 45-48 hores d’hibridació. Tot i que a aquest temps se li ha d’afegir les dues hores necessàries per a l’anàlisi de cada corpuscle, aquest protocol permet obtenir de resultats el dia +3 o +4 (considerant com a dia 0 el dia de la punció fol.licular). Així, la transferència embrionària es pot fer el dia +4, dia en què s’aconsella la transferència després de qualsevol PGD (Gianaroli et al. 1999b).

181 Aquesta estratègia permetria analitzar la totalitat de cromosomes en un PGD i evitaria la congelació dels embrions, requisit imprescindible per fer l’anàlisi de CGH en blastòmers biòpsiats a dia +3 (Voullaire et al. 2002; Wells et al. 2002; Wilton et al. 2003a).

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6. CONCLUSIONS

1. La tècnica de CGH permet analitzar la totalitat dels cromosomes en cèl.lules aïllades. Tanmateix, alteracions en els blocs heterocromàtics, regions centromèriques i telomèriques, així com aneuploïdies dels cromosomes 17, 19 i 22 han de ser considerades amb precacució ja que són regions propenses a donar falsos positius.

2. La CGH és capaç de detectar tant errors de cromosoma com de cromàtida, encara que molt sovint no permet diferenciar entre aquests errors. La desviació dels perfils en la CGH aplicada a cèl.lules aïllades és més pronunciada en els casos de pèrdues que de guanys.

3. L’eficiència i la fiabilitat de la CGH aplicada a l’anàlisi de cèl.lules aïllades és major que la d’altres tècniques de citogenètica molecular basades en la fixació del material cromosòmic en portaobjectes, ja que aquesta tècnica permet avaluar de manera acurada la incidència no tan sols de les hiperhaploïdies sinó també de les hipohaploïdies de pràcticament qualsevol cromosoma.

4. L’anàlisi d’ambdues cèl.lules, 1CP i MII, mitjançant la combinació de la CGH amb la FISH, l’SKY i el cenM-FISH, que ha estat aplicat en oòcits per primer cop en el present treball, és útil per determinar la incidència global d’aneuploïdies en els oòcits estudiats, per confirmar els resultats obtinguts, així com per investigar els mecanismes implicats en l’origen de les aneuploïdies: la no-disjunció, la separació precoç de cromàtides i el mosaïcisme gonadal.

5. La incidència d’aneuploïdia depèn de l’edat materna ja que s’han trobat diferències significatives en els oòcits dels dos grups d’edat analitzats. Tanmateix, la incidència d’aneuploïdia no depèn de l’origen dels oòcits ja que el nombre d’anomalies cromosòmiques no són significativament diferents en analitzar oòcits madurats in vitro o oòcits descartats per causa d’una fallada de fertilització.

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6. L’anomalia més freqüent en la primera divisió meiòtica no és la no-disjunció de cromosomes sinó la divisió precoç de cromàtides germanes, amb una afectació preferent dels cromosomes més petits.

7. S’ha demostrat la importància de l’anàlisi de la totalitat de cromosomes en el PGD-AS ja que s’han detectat aneuploïdies de pràcticament tots els cromosomes estudiats.

8. L’anàlisi amb FISH emprant sondes per als 9 cromosomes analitzats rutinàriament en el PGD-AS hauria diagnosticat incorrectament com a normals d’un 25% a un 30% dels oòcits aneuploides. A més, el PGD-AS utilitzant FISH exclou l’anàlisi de més de la meitat dels cromosomes de la cèl.lula, alguns d’ells (cromosomes 1, 4 i 19), afectats freqüentment per errors i possiblement implicats en les pèrdues embrionàries primerenques i en les fallades d’implantació de la majoria de pacients.

9. La CGH pot ser utilitzada per detectar segregacions desequilibrades de translocacions maternes en el PGD de translocacions quan els fragments implicats siguin majors de 10-40 Mb. Aquesta estratègia permet detectar anomalies no tan sols dels cromosomes reorganitzats, sinó també de la resta de cromosomes i minimitzar d’aquesta manera la transferència d’embrions anòmals incorrectament diagnosticats com a normals.

10. El protocol posat a punt i optimitzat en el nostre laboratori ha estat validat i és apte per ser aplicat en el PGD mitjançant l’anàlisi del 1CP. Aquest protocol de PGD és compatible amb la transferència embrionària el dia +4.

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